The Marshall W. Nirenberg Papers

Summary of work for this project as indicated on the report: "Our work on muscarinic acetylcholine receptors focuses on
two fundamental questions: (1) How is binding of acetylcholine to receptors converted into an intracellular response? (2)
What factors regulate receptor concentrations in the membrane? A binding assay employing tritiated quinuclidinyl benzilate
permits us to investigate properties of receptor binding sites and to measure receptor levels in cultured neuroblastoma and
hybrid cells under systematically varied conditions."

Objectives: The objectives of our work have been (1) to characterize the nature of ligand interactions with muscarinic receptors,
and (2) to assess various factors likely to affect receptor concentrations. Although most methodology involved has been straightforward,
our experiments required the custom labeling and purification of muscarinic ligands as commercially available materials were
unsuitable due to low specific activity.

Major Findings: The principal findings are:

(1) Excitatory and inhibitory muscarinic receptors for ligands which do not differ pharmacologically.

(2) The affinity of receptors for acetylcholine is affected by the ionic composition of the medium.

(3) Antagonists and agonists differ strikingly in the way that they interact with the receptor. Antagonist binding to receptor
follows the law of mass action whereas agonist binding does not. When analyzed on Hill plots, agonist interactions with receptor
exhibit negative cooperativity that may be related to receptor desensitization.

(4) The receptor concentration is greatly reduced by exposure of cultured cells to carbamylcholine or other receptor activators.
The [receptor-agonist] complex is degraded more rapidly than the free receptor.

(5) Muscarinic excitatory and inhibitory acetylcholine receptors have been solubilized and have been characterized partially.

Significance to Biomedical Research: This work with a model system suggests that, in the normal nervous system, receptor
levels, which ultimately influence the pattern of behavior of neural nets, are regulated by synaptic transmission. Some of
the events which result from the interaction of acetylcholine with muscarinic acetylcholine receptors have been defined.
Two regulatory processes were found which depend upon acetylcholine. One regulates receptor activity, the other regulates
receptor concentration.